The h subunit of eIF3 promotes reinitiation competence during translation of mRNAs harboring upstream open reading frames

Abstract

Upstream open reading frames (uORFs) are protein coding elements in the 5' leader of messenger RNAs. uORFs generally inhibit translation of the main ORF because ribosomes that perform translation elongation suffer either permanent or conditional loss of reinitiation competence. After conditional loss, reinitiation competence may be regained by, at the minimum, reacquisition of a fresh methionyl-tRNA. The conserved h subunit of Arabidopsis eukaryotic initiation factor 3 (eIF3) mitigates the inhibitory effects of certain uORFs. Here, we define more precisely how this occurs, by combining gene expression data from mutated 5' leaders of Arabidopsis AtbZip11 (At4g34590) and yeast GCN4 with a computational model of translation initiation in wild-type and eif3h mutant plants. Of the four phylogenetically conserved uORFs in AtbZip11, three are inhibitory to translation, while one is anti-inhibitory. The mutation in eIF3h has no major effect on uORF start codon recognition. Instead, eIF3h supports efficient reinitiation after uORF translation. Modeling suggested that the permanent loss of reinitiation competence during uORF translation occurs at a faster rate in the mutant than in the wild type. Thus, eIF3h ensures that a fraction of uORF-translating ribosomes retain their competence to resume scanning. Experiments using the yeast GCN4 leader provided no evidence that eIF3h fosters tRNA reaquisition. Together, these results attribute a specific molecular function in translation initiation to an individual eIF3 subunit in a multicellular eukaryote.

FIGURE 1.

In eif3h mutant plants, 43S complexes lack eIF3h. (A) Wild-type eIF3h and truncated eIF3h expressed in eif3h-1 mutant plants were visualized by immunoblotting with anti-eIF3h antibody. (*) Crossreacting protein. (B) Sucrose gradients from wild-type and eif3h mutant plants were examined for 18S ribosomal RNAs to identify 40S subunits using electrophoresis and ethidium bromide staining. Corresponding protein extracts were examined for eIF3 subunits by SDS-PAGE and immunoblotting. (Arrows) Topmost fractions containing 40S and 60S subunits.

FIGURE 2.

eIF3h-dependent translational regulation of the AtbZip11 5′ leader. (A) (Top) Schematic of the FLUC reporter coding region fused to original and mutated 5′ leaders of AtbZip11 (590 nt long). Only the four uORFs are drawn to scale (white boxes; 18, 42, 5, and 19 amino acids, respectively). (Middle) Transient expression data from 10-d-old wild-type and eif3h mutant seedlings. The efficiency of translation was calculated as the activity ratio of FLUC versus Renilla luciferase (RLUC) as a co-transformed reference gene. Bars, standard error. (*) P < 0.002 for wild-type (WT) versus eif3h in a two-sided Students t-test. (Bottom) Respective transcript levels of FLUC reporter and translation elongation factor 1α (EF1α) as a control were compared by RT-PCR. (B) Turnover of AtbZip11-FLUC mRNA expressed in stable transgenic plants (Kim et al. 2007) was measured by RT-PCR after blocking transcription with 0.1 mg/mL cordycepin. EF1α served as a control. Neither mRNA was labile over this time course. (C) Translation of the AtbZip11-FLUC reporter construct with the default 3′ UTR from cauliflower mosaic virus was similar to that with the native AtbZip11 3′ UTR. (*) P < 0.002 (n = 5). (D) Undertranslation of the AtbZip11 5′ leader in the eif3h mutant in Arabidopsis protoplasts transformed with in vitro transcripts. “Spacer” is an example for a uORF-less leader that is unaffected by eif3h.

FIGURE 3.

Contribution of individual uAUGs to translational repression and eIF3h-dependent translation of AtbZip11-FLUC. (A) Single uAUGs were eliminated by site-directed mutagenesis. Note the anti-inhibitory effect of uORF 1 (also see C). Error bars represent standard error (n = 8–11). Data on the original and uORF-less leaders are included from Figure 2A for comparison. (*) Statistical significance at P < 0.01, (**) P < 0.002. (B) The combination of uORFs 2 and 3 caused a robust dependence on eIF3h, in contrast to uORFs 1 and 4 together (n = 8–12). eIF3h dependence of uORF2 alone was also significant but that of uORF3 alone was not. (*) Statistical significance at P < 0.01. (Right panel) AtbZip-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR. (C) A pairwise comparison demonstrating the anti-inhibitory effect of uORF1 in the AtbZip11 leader. Note that leaders harboring uORF1 are still eIF3h-dependent. Bars, standard error (n = 9–22). (*) Statistical significance at P < 0.04, (**) P < 0.01.

FIGURE 4.

uAUG recognition. (A) The uAUGs of the AtbZip11 uORFs were fused directly to FLUC while retaining the start codon context up to the +4 position as well as their distance from the transcription start site as shown. Note that all uAUGs appear to be recognized in an eIF3h-independent fashion (n = 7–14). Experimental conditions are as in Figure 2. (B) uAUG-FLUC fusion constructs to examine the (re)initiation potential at individual uAUGs downstream from uORF1. Bars, standard error (n = 5–6). (*) Statistical significance at P < 0.02.

FIGURE 5.

Requirement for eIF3h for efficient reinitiation. (A) To delineate leaky scanning across uORFs 2 and 3, all in-frame stop codons were eliminated by point mutations, thus causing them to overlap the main FLUC ORF by 44 and 45 amino acids, respectively (2a,2b,3 ovlp). Residual expression of FLUC is attributed to ribosomes that have leaky-scanned uORFs 2a, 2b, and 3. (B) The effect of eIF3h on reinitiation downstream from uORF 2a,2b,3 was tested by shortening the intercistronic spacer length in the AtbZip11 leader by deletion from the 3′ end of the spacer. uORFs 2a, 2b, and 3 were placed in a strong Kozak context (₋₃aaaAUGg₊₄) to minimize the effect of leaky scanning. Error bars, standard error (n = 5–8). (*) Statistical significance at P < 0.002. (C) Inhibition of translation by an ATF4-like (Harding et al. 2000) overlap-uORF downstream from uORF2 demonstrates consistent reinitiation in wild type but marginal reinitiation in eif3h. A uORF (gray box) in a strong Kozak context (A₋₃AAAUGG₊₄) was created starting 60 nt upstream of the FLUC ORF to overlap the main ORF. (*) AUGs in a strong context. Bars, standard error (n = 8–12). AtbZip-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR.

FIGURE 6.

Computer simulation of the translational defect in eif3h mutant plants. (A) Model parameters. (Gray boxes) ORFs. Spanning bars indicate the range over which a given term applies. (B–F) Distributions of parameter estimates. x-axis length reflects the manually set boundaries of possible parameter estimates. y-axis counts indicate the number of times out of 100 trials that a parameter fell into one of 20 x-axis bins. (B) Loss rate of reinitiation competence (k₁) as a function of uORF length (u [nt]). (C) Regaining of reinitiation competence (k₂) as a function of intercistronic spacer length (s [nt]). (D) Probability of AUG recognition in a strong context (p_cs). (E) AUG recognition in a weak context (p_cw). (F) Escape from attenuation upon translation of uORF2b peptide (p_2b). (G) Scatter plot illustrating the match between model output using MLEs (x-axis) and experimental data (y-axis, ± one standard error) for all 21 AtbZip11 5′ leader constructs in wild-type (dark green) and eif3h plants (red). (Light green symbols) Predicted expression values using model parameters culled from the literature (see Table 1).

FIGURE 7.

Stimulation of translation across uORFs 2 and 3 by eIF3h is not dependent on the uORF peptide sequences. Two independent frameshift mutations were introduced into uORF2a/2b to change the sequence of the encoded peptide while retaining its native length and also the flanking sequences. For uORF3, point mutations were introduced to change three out of its five amino acids. The frameshift data (wild type versus eif3h) were statistically significant by paired t-test (P < 0.002).

FIGURE 8.

Translation initiation on the 5′ leader from yeast GCN4 in Arabidopsis, and microarray meta-analysis. (A) The schematic illustrates the predictions of two contrasting hypotheses concerning the molecular function of eIF3h (after Hinnebusch 2005). uORF4 is shaded (gray) for emphasis. If eIF3h supported resumption of scanning, few ribosomes would scan the intercistronic spacer in the eif3h mutant, but those that do would easily acquire a fresh ternary complex (TC) and initiate at the inhibitory uORF4. Hence, adding uORF4 to uORF1 will reduce FLUC translation to similar degrees in wild type and eif3h (bars on the right symbolize predicted FLUC expression levels). In contrast, if the eif3h mutation delayed TC acquisition, some mutant ribosomes would leaky-scan past uORF4 and thus reach the main FLUC ORF. Meanwhile, wild-type ribosomes, having acquired their TC early, will be preferentially intercepted by uORF4, resulting in a reversal of the eIF3h dependence (bars on right). (B) GCN4 5′ leader sequences tested. The version with four uORFs is the original. (*) uAUGs in a strong context. (C) Expression data for the constructs shown in panel B. Error bars, standard error (n > 4). (*) Statistical significance at P < 0.05. Where shown, GCN4-FLUC and endogenous EF1α mRNA levels were estimated by RT-PCR. (D) The translation state of Arabidopsis mRNAs corresponding to the scheme outlined at the top was mined from polysome microarray data (Kim et al. 2007) and displayed for wild type and eif3h mutant. Translation state is defined as the log(2) of the ratio of polysomal (PL) to nonpolysomal (NP) mRNA signal. Note that the translation state is equal or lower in eif3h than in wild type, as predicted by the resumption-of-scanning hypothesis.